PROJECT SUMMARY Post-surgical pain causes significant suffering to patients and an over-reliance on opioids for pain reduction. However, opioid analgesics cause severe side effects and a risk of accidental death from overdose. Unfortunately, effective non-opioid pain therapy remains lacking. We aim to determine if human cell and tissue product (HCT/P), such as amniotic membrane and umbilical cord, can be developed for use as a novel and safe treatment of post-surgical pain. In Aim 1, we will first prove the concept by demonstrating the efficacy, receptor mechanisms, and safety of HCT/P for inhibiting post-surgical pain in murine models. Our preliminary studies showed that intraoperative treatment with HCT/P reduced the development of post-surgical pain in rats. In Aim 2, we will demonstrate that HCT/P targets immune cells as an indirect mode of anti-pain action. We hypothesize that HCT/P promotes regenerative wound healing by suppressing immune cell recruitment and inducing anti-inflammatory effects. Our recent studies suggested that the heavy chain-hyaluronic acid-pentraxin 3 complex (HC-HA/PTX3) is a biologically active component uniquely present in HCT/P. We will further explore how HC-HA/PTX3 polarizes macrophages differentiated from human THP-1 cells to an M2 phenotype and whether HC-HA/PTX3 inhibits the activation of human LAD2 mast cell and release of pro-inflammatory cytokines. In Aim 3, we will examine the targeting of primary sensory neurons as the direct mode of anti-pain action by HCT/P and HC-HA/PTX3. We will test whether HC-HA/PTX3 reduces intrinsic membrane excitability and inhibits membrane ion channels in dorsal root ganglion neurons. Furthermore, we will investigate whether the neuronal inhibition by HC-HA/PTX3 involves a novel mechanism that depends on CD44-mediated cytoskeletal rearrangement. To achieve these goals, we will combine the expertise of preclinical and translational pain researchers from Johns Hopkins University School of Medicine and the GMP manufacturing of HCT/P at TissueTech, Inc. This study will provide important rationales for developing HCT/P-based bioceuticals as a viable non-opioid treatment of post-surgical pain. Such drugs will act not only directly, by dampening neuronal excitation, but also indirectly, by changing the wound environment and orchestrating regenerative healing. Through the complementary dual mode of action, we envision that birth HCT/P may lead to an optimal therapeutic effect with lasting pain relief and a potential ?cure? of post-surgical pain.